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Abstract：
Rationale: TOX is a DNA-binding factor required for the development of multiple immune cells and the formation of lymph nodes. However, the temporal regulation mode of TOX on NK cell development and function needs to be further explored. Methods: To investigate the role of TOX in NK cells at distinct developmental phases, we deleted TOX at the hematopoietic stem cell stage (Vav-Cre), NK cell precursor (CD122-Cre) stage and late NK cell developmental stage (Ncr1-Cre), respectively. Flow cytometry was used to detect the development and functional changes of NK cell when deletion of TOX. RNA-seq was used to assess the differences in transcriptional expression profile of WT and TOX-deficient NK cells. Published Chip-seq data was exploited to search for the proteins directly interact with TOX in NK cells. Results: The deficiency of TOX at the hematopoietic stem cell stage severely retarded NK cell development. To a less extent, TOX also played an essential role in the physiological process of NKp cells differentiation into mature NK cells. Furthermore, the deletion of TOX at NKp stage severely impaired the immune surveillance function of NK cells, accompanied by down-regulation of IFN-γ and CD107a expression. However, TOX is dispensable for mature NK cell development and function. Mechanistically, by combining RNA-seq data with published TOX ChIP-seq data, we found that the inactivation of TOX at NKp stage directly repressed the expression of Mst1, an important intermediate kinase in Hippo signaling pathway. Mst1 deficient at NKp stage gained the similar phenotype with Toxfl/flCD122Cre mice. Conclusion: In our study, we conclude that TOX coordinates the early mouse NK cell development at NKp stage by maintaining the expression of Mst1. Moreover, we clarify the different dependence of the transcription factor TOX in NK cells biology.
摘要：
原理：TOX是多种免疫细胞发育和淋巴结形成所需的DNA结合因子。然而,TOX对NK细胞发育和功能的时间调控模式有待进一步探讨。方法：探讨TOX在不同发育阶段NK细胞中的作用，我们在造血干细胞阶段删除了TOX（Vav-Cre），NK细胞前体（CD122-Cre）阶段和晚期NK细胞发育阶段（Ncr1-Cre），分别。流式细胞术检测TOX缺失时NK细胞的发育和功能变化。RNA-seq用于评估WT和TOX缺陷型NK细胞的转录表达谱的差异。利用公开的Chip-seq数据来搜索与NK细胞中的TOX直接相互作用的蛋白质。结果：造血干细胞阶段TOX的缺乏严重阻碍了NK细胞的发育。在较小程度上，TOX在NKp细胞分化为成熟NK细胞的生理过程中也起着重要作用。此外，在NKp阶段TOX的缺失严重损害了NK细胞的免疫监视功能，伴随着IFN-γ和CD107a表达的下调。然而,TOX对于成熟的NK细胞发育和功能是必不可少的。机械上,通过将RNA-seq数据与已发布的TOXChIP-seq数据相结合，我们发现TOX在NKp阶段的失活直接抑制了Hippo信号通路中重要的中间激酶Mst1的表达。在NKp阶段缺乏的Mst1获得了与Toxfl/flCD122Cre小鼠相似的表型。结论：在我们的研究中，我们得出结论，TOX通过维持Mst1的表达来协调NKp阶段早期小鼠NK细胞的发育。此外，我们阐明了转录因子TOX在NK细胞生物学中的不同依赖性。
